The present invention relates to the field of cell culture of human keratinocyte precursor and dermal fibroblast cells. In addition, the present invention also relates to the use of cultured keratinocyte precursor cells in the repair of skin defects by skin grafting procedures.
The healing of skin defects progresses through three general phases: (i) inflammation, (ii) wound cell migration and mitosis, and (iii) extracellular matrix production and remodeling. The ordered sequence of these events is thought to be orchestrated by interactions among cells, growth factors, and extracellular matrix proteins. A crucial step of skin wound healing is epidermal regeneration (i.e., re-epithelialization). Besides interfollicular epidermal keratinocytes from the wound edges, the outer root sheath (ORS) cells from residual hair follicles also contribute to this process. See e.g., Eisen, et al., 1955. J. Invest. Dermatol. 15: 145-155. The ORS of hair follicles is comprised largely of undifferentiated keratinocytes that encompass the cylindrical structures of the hardened inner root sheath and the hair shaft. See e.g., Montagna and Parakkal, 1974. pp. 172-258. In: The Structure and Function of Skin (Academic Press New York, N.Y. Recent literature has also indicated that ORS cells are at a lower level of commitment to differentiation than the basal interfollicular keratinocytes (see e.g., Coulombe, et al., 1989. J Cell Biol. 109: 2295-2312; Limat, et al., 1991. Exp. Cell Res. 194: 218-227; Limat, et al., 1994. Cell Tissue Res. 275: 169-176 1994), and label-retaining cells have been detected in the animal as well as the human ORS region near the bulge area which possibly represent stem cells for skin epithelial tissues. See e.g., Cotsarelis, et al., 1990. Cell 61: 1329-1337; Kobayashi, et al., 1993. Proc. Natl. Acad. Sci. USA 90: 7391-7395; Yang, et al., 1993. J. Invest. Dermatol. 105: 14-21; Rochat, et al., 1994. Cell 76: 1073-1076; Moll, 1995. J. Invest. Dermatol. 105: 14-21. Additionally, human ORS cells which are isolated from plucked anagen scalp hair follicles can be expanded extensively in vitro. See e.g., Weterings, et al., 1981. Brit. J. Dermatol. 104: 1-5; Limat and Noser, 1986. J. Invest. Dermatol. 87: 485-488; Imcke, et al., 1987. J. Am. Acad. Dermatol. 17: 779-786; Limat, et al., 1989. J. Invest. Dermatol. 92: 758-762. Under conventional submerged culture conditions, ORS cells resemble interfollicular epidermal keratinocytes by both morphologic and biochemical (e.g., keratin profiles) criteria. See e.g., Stark, et al., 1987. Differentiation 35: 236-248; Limat, et al., 1989. J. Invest. Dermatol. 92: 758-762; Limat, et al., 1991. Ann. N.Y. Acad. Sci. 642: 125-147. In organotypic co-cultures with human dermal fibroblasts (i.e., under conditions mimicking the epidermal environment), ORS cells with respect to histological, immunohistological, ultrastructural and biochemical criteria develop a stratified epithelium reminiscent of regenerating epidermis. See e.g., Lenoir, et al., 1988. Dev. Biol. 130: 610-620; Limat, et al., 1991. Exp. Cell Res. 194: 218-227; Limat, et al., 1991. Ann. N.Y. Acad. Sci. 642: 125-147. If such organotypic cultures are grafted onto nude mice, ORS cells form a regular neo-epidermis that is under homeostatic control. See e.g., Limat, et al., 1995. Transplantation 59: 1032-1038. Thus, human ORS cells are of considerable interest for clinical application.
In the previous decade, interest has focused on the use of cultured epithelial cells for wound coverage. First, sheets of cultured autologous interfollicular keratinocytes were grafted successfully on acute wounds, mainly in the treatment of larger third degree burns (see e.g., O""Connor, et al., 1981. Lancet 1: 75-78; Compton, et al., 1989. Lab. Invest. 60: 600-612) but also of epidermolysis bullosa (see e.g., Carter, et al., 1987. J. Am. Acad. Dermatol. 17: 246-250), pyoderma gangrenosum (see e.g., Dean, et al., 1991. Ann. Plast. Surg. 26: 194-195; Limova and Mauro, 1994. J. Dermatol. Surg. Oncol. 20: 833-836), and wounds after excision of giant congenital nevi (see e.g., Gallico, et al., 1989. J. Plast. Reconstr. Surg. 84: 1-9) or separation of conjoined twins (see e.g., Higgins, et al., 1994. J. Royal Soc. Med. ??:108-109).
In contrast to the treatment of such acute wounds, the grafting of chronic wounds (e.g., leg ulcers) with cultured keratinocytes has been much less successful. Allografts do not result in a permanent xe2x80x9ctakexe2x80x9d (see e.g., Fabre, 1991. Immunol. Lett. 29: 161-166) and thus may be classified as a xe2x80x9c . . . quite effective but expensive biological dressingxe2x80x9d. See Phillips, et al., 1989. J Am. Acad. Dermatol. 21: 191-199. A reproducible, major definite xe2x80x9ctakexe2x80x9d of autologous keratinocyte grafted by various modalities including: sheets of submerged keratinocyte cultures consisting of only a few, noncornified cell layers (Hetton, et al., 1986. J. Am. Acad. Dermatol. 14: 399-405; Leigh and Purkis, 1986. Clin. Exp. Dermatol. 11: 650-652; Leigh, et al., 1987. Brit. J. Dermatol. 117: 591-597; Harris, et al., 1993. Clin. Exp. Dermatol. 18: 417-420), trypsinized single cells attached to collagen-coated dressings (Brysk, et al., 1991. J. Am. Acad. Dermatol. 25: 238-244), skin equivalents (Mol, et al., 1991. J. Am. Acad. Dermatol. 24: 77-82, 1991) has yet to be convincingly documented within the scientific literature. The same lack of quantitative findings also holds true for various reports on the grafting of freshly isolated, autologous interfollicular keratinocytes (Hunyadi, et al., 1988. J. Dermatol. Surg. Oncol. 14: 75-78) or ORS cells (Moll, et al., 1995. Hautarzt 46: 548-552) fixed to the wound bed by the use of a fibrin glue. However, it should be noted that the disadvantages of the bovine serum used during cultivation of the keratinocytes may contribute to reduced xe2x80x9ctakexe2x80x9d rate, due to the fact that it resists in keratinocytes. See e.g., Johnson, et al., 1990. J. Burn Care Rehab. 11: 504-509.
Prior to the disclosure of the present invention herein, the standard methodology for the generation of a primary culture of basal keratinocytes consisted of the plucking of an anagenic (i.e., growing hair shaft) hair followed by a careful microscopic dissection to remove the hair bulbs and the infundibular hair shaft. The resulting outer root sheath (ORS) was then placed on the culture insert for initiation of the primary keratinocyte culture. However, numerous subsequent studies (approximately 200), wherein the anagenic hair was placed directly on the culture insert without performing the initial micro-dissection to remove the hair bulbs and the infundibular hair shaft, have demonstrated that such tedious and time-consuming dissection of the plucked anagenic hair was not required. This has served to markedly simplify the handling process, reduce the risk for contamination, and resulted in more efficient initiation of keratinocyte cell plating.
Accordingly, it is an object of the present invention to provide improved and simplified methods for the generation of keratinocytes or keratinocyte precursors from outer root sheath cells (ORS cells) in fully defined culture conditions for the treatment of various types of skin defects (e.g., chronic wounds such as leg ulcers, diabetic ulcers, pressure sores, and the like) in both humans and animals. In addition to their use in the treatment of wounds, keratinocytes may also be used in plastic and cosmetic surgery, or whenever there is a demand for such skin support (e.g., post operative following the removal of tattoos, naevi, skin cancer, papillomas, after amputation, in sex transformation or re-virgination, and the like).
These aforementioned objectives are accomplished by explantation and culture of plucked, anagenic or growing hairs in toto upon microporous membranes carrying human fibroblast feeder cells at their under-surface. In such primary cultures, large numbers of ORS cells can be easily and repeatedly obtained, irrespective of the donor""s chronological age. Such ORS cells may be used for the subsequent preparation of dermal or epidermal equivalents or kept frozen and stored in order to use them at a later time point.
The subsequent preparation of dermal or epidermal equivalents is achieved by the xe2x80x9cseedingxe2x80x9d of these ORS cells upon a modified, microporous membranes carrying fibroblast feeder cells (most preferably growth-arrested/limited human fibroblast xe2x80x9cfeeder cellsxe2x80x9d) at their under-surface. During culture, these ORS cells undergo tissue differentiation which has been demonstrated to be similar to that of normal epidermis. This finding is most probably due to a large compartment of proliferating cells. The modified culture conditions which are disclosed herein are important for the successful treatment of chronic wounds with epidermal equivalents generated in vitro from autologous ORS cells.
A further object of the present invention to provide improve culture systems for ORS-derived keratinocytes by adhering the anagenic hair onto a polymeric microporous membrane coated with one or more molecules of extracellular matrix origin. These improved cultures of ORS cells, designated as dermal equivalents or epidermal equivalents, may be used to treat skin defects, especially chronic wounds.
Yet another object of the present invention to produce dermal or epidermal equivalents using a reduced concentration of allogenic or homologous serum. This greatly mitigates the risk of disease transmission, for example, by clinical use of blood products, by the use of autologous or homologous human serum and substances derived or released from blood components (e.g., blood platelets) for supplements in in vitro culturing steps.
A further object of the present invention is a methodology which ameliorates the probability of mechanical damage (e.g., separation of the various constituent layers) of the dermal or epidermal equivalents during transport prior to transplantation.
The clinical advantages of the methodology of the present invention, as compared to grafting techniques of chronic wounds which have been previously utilized, include, but are not limited to: noninvasiveness (so that the cells are available repeatedly), the lack of need for surgical facilities or anesthesia during the grafting procedure, and a short immobilization period of only 2 hours is required following the grafting procedure.